Development of a rapid bacterial identification method based on direct mass spectrometric metabolic profiling

该项目旨在开发新的工具，用于识别各种微生物，包括导致从普通感冒到血液感染等广泛疾病的细菌。准确了解哪种细菌与疾病有关是非常重要的，因为选择适当的药物在很大程度上取决于它。同样，在公共卫生或食品安全的情况下，细菌污染的正确分类有助于确定其来源和消除污染。目前，含有细菌细胞的样本被收集并送往实验室。微生物学家在含有特殊营养物质的培养皿中培养细菌。根据该菌可利用的营养物质种类和多种化学试验结果，初步鉴定了该菌。如果有必要进行适当的分类，则从细菌细胞中提取核酸，并确定其碱基对序列，这有助于明确识别。所有这些过程都很耗时，在传染病和水传播疾病爆发的情况下，这大大延迟了有效的干预。 这项研究的目的是开发一种替代的、更快的细菌鉴定技术。质谱是一种能够测量分子重量的分析技术，也可以同时选择性地检测数百种不同的分子。我们计划使用这种成熟的技术来观察细菌细胞的一些特殊构建模块。这项研究的一个新颖之处在于，质谱仪不是以传统的方式使用的，包括在分析之前对细菌细胞进行冗长的准备，而是简单地加热细胞，并使用质谱法分析细胞沸腾时形成的带电分子。通过简单加热进行快速质谱分析的想法已经应用于手术中，其中癌症组织以类似的方式被识别。在拟议的项目过程中，我们计划采用该技术（快速蒸发电离质谱法; REIMS）分析实验室中生长的细菌，并直接分析含有细菌制剂的液体样品（从池塘水到血液）。我们计划建立一个大型的细菌光谱指纹库，它将被用作基于计算机的搜索算法的训练集。该方法，数据库和算法将共同实现在比当前常规短得多的时间内明确识别细菌。此外，拟议的研究可能会导致一种方法，其中细菌在其自然环境中直接识别（例如，在尿液中用于尿路感染），而无需在实验室中培养数小时或数天。

The project is aimed at the development of new tools for the identifications of various microorganisms including bacteria causing a wide range of diseases from common cold to bloodstream infections. Knowing exactly which type of bacterium is involved in a disease is very important, since the choice of appropriate medication largely depends on it. Likewise, in case of public health or food safety, the correct classification of bacterial contaminations helps with the identification of their source and elimination of the contamination. Currently, samples containing bacterial cells are collected and sent to laboratories. Microbiologists grow the bacteria in Petri-dishes containing special nutrients. Based on the types of nutrients the bacteria can use and the results of multiple chemical tests, the bacterium is tentatively identified. If proper classification is necessary, nucleic acids are extracted from the bacterial cells, and their base-pair sequence is determined, which helps in the unambiguous identification. All of these processes are time consuming, which considerably delays the efficient intervention both in case of infectious diseases and in case of a waterborne disease outbreak. The purpose of the proposed research is to develop an alternative, much faster technique for the identification of bacteria. Mass spectrometry is an analytical technique capable of the measurement of the weight of molecules, and also the selective detection of hundreds of different molecules at the same time. We plan to use this well-established technique for looking at some special building blocks of bacterial cells. One novel aspect of the research is that mass spectrometers are not used in the traditional way, including a lengthy preparation of bacterial cells prior to analysis, but the cells are simply heated up, and electrically charged molecules formed on the boiling of cells are analysed using mass spectrometry. The idea of rapid mass spectrometric analysis by using simply heat has already been applied in case of surgery, where cancer tissue is identified in a similar way. In course of the proposed project we plan the adopt this technology (Rapid Evaporative Ionization Mass spectrometry; REIMS) for the analysis of bacteria grown in the laboratory and also for the direct analysis of liquid samples (ranging from pond water to blood) containing bacterial agents. We plan to build a large library of the spectroscopic fingerprints of the bacteria, which will be used as a training set for computer based search algorithms. The method, the database and the algorithm together will enable the unambiguous identification of bacteria in considerably shorter timeframe than the current routine. Furthermore, the proposed research can potentially lead to an approach, where bacteria are directly identified in their natural environment (e.g. in urine for a urinary infection) without growing them in the laboratory for several hours or days.